CN116920173B - PDA-D-amino-TNT composite coating for dental implant and preparation method thereof - Google Patents
PDA-D-amino-TNT composite coating for dental implant and preparation method thereof Download PDFInfo
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- CN116920173B CN116920173B CN202310766988.1A CN202310766988A CN116920173B CN 116920173 B CN116920173 B CN 116920173B CN 202310766988 A CN202310766988 A CN 202310766988A CN 116920173 B CN116920173 B CN 116920173B
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- 238000000576 coating method Methods 0.000 title claims abstract description 24
- 239000011248 coating agent Substances 0.000 title claims abstract description 22
- 239000002131 composite material Substances 0.000 title claims abstract description 16
- 239000004053 dental implant Substances 0.000 title claims abstract description 16
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 51
- 229920001184 polypeptide Polymers 0.000 claims abstract description 48
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 48
- 239000000243 solution Substances 0.000 claims abstract description 43
- 238000012360 testing method Methods 0.000 claims abstract description 42
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- 238000000034 method Methods 0.000 claims abstract description 18
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 15
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- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 11
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 claims abstract description 10
- 230000004048 modification Effects 0.000 claims abstract description 9
- 238000012986 modification Methods 0.000 claims abstract description 9
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- 238000003491 array Methods 0.000 claims abstract description 8
- 239000011229 interlayer Substances 0.000 claims abstract description 6
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 42
- 238000006243 chemical reaction Methods 0.000 claims description 34
- 239000011347 resin Substances 0.000 claims description 28
- 229920005989 resin Polymers 0.000 claims description 28
- 229910052757 nitrogen Inorganic materials 0.000 claims description 22
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Natural products CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 claims description 20
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 19
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 19
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 18
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 18
- 239000010936 titanium Substances 0.000 claims description 17
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- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 claims description 13
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 6
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- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 6
- -1 benzotriazol-N, N, N ', N' -tetramethyl urea hexafluorophosphate ester Chemical class 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 4
- 239000004473 Threonine Substances 0.000 claims description 4
- 238000010511 deprotection reaction Methods 0.000 claims description 4
- 239000003792 electrolyte Substances 0.000 claims description 4
- 238000001291 vacuum drying Methods 0.000 claims description 4
- QWXZOFZKSQXPDC-NSHDSACASA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C)C(O)=O)C3=CC=CC=C3C2=C1 QWXZOFZKSQXPDC-NSHDSACASA-N 0.000 claims description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 3
- 230000032683 aging Effects 0.000 claims description 3
- 235000011089 carbon dioxide Nutrition 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
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- 239000000706 filtrate Substances 0.000 claims description 3
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- 230000014759 maintenance of location Effects 0.000 claims description 3
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 claims description 3
- 239000012074 organic phase Substances 0.000 claims description 3
- 238000004806 packaging method and process Methods 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
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- 238000000746 purification Methods 0.000 claims description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 3
- 238000004007 reversed phase HPLC Methods 0.000 claims description 3
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- 238000001308 synthesis method Methods 0.000 claims description 3
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims description 2
- 229910017855 NH 4 F Inorganic materials 0.000 claims 1
- 230000002194 synthesizing effect Effects 0.000 claims 1
- 239000007943 implant Substances 0.000 description 19
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 16
- 230000000844 anti-bacterial effect Effects 0.000 description 8
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- 241000894006 Bacteria Species 0.000 description 4
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
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- LZOLWEQBVPVDPR-VLIAUNLRSA-N (2s,3r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]butanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H]([C@H](OC(C)(C)C)C)C(O)=O)C3=CC=CC=C3C2=C1 LZOLWEQBVPVDPR-VLIAUNLRSA-N 0.000 description 2
- VYMPLPIFKRHAAC-UHFFFAOYSA-N 1,2-ethanedithiol Chemical compound SCCS VYMPLPIFKRHAAC-UHFFFAOYSA-N 0.000 description 2
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- LTYMSROWYAPPGB-UHFFFAOYSA-N diphenyl sulfide Chemical compound C=1C=CC=CC=1SC1=CC=CC=C1 LTYMSROWYAPPGB-UHFFFAOYSA-N 0.000 description 2
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- 238000007710 freezing Methods 0.000 description 2
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- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 2
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- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 description 1
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- HSFWRNGVRCDJHI-UHFFFAOYSA-N alpha-acetylene Natural products C#C HSFWRNGVRCDJHI-UHFFFAOYSA-N 0.000 description 1
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- LDDQLRUQCUTJBB-UHFFFAOYSA-N ammonium fluoride Chemical compound [NH4+].[F-] LDDQLRUQCUTJBB-UHFFFAOYSA-N 0.000 description 1
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- 125000002534 ethynyl group Chemical group [H]C#C* 0.000 description 1
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- 210000004872 soft tissue Anatomy 0.000 description 1
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- 210000001519 tissue Anatomy 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/28—Materials for coating prostheses
- A61L27/30—Inorganic materials
- A61L27/306—Other specific inorganic materials not covered by A61L27/303 - A61L27/32
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/28—Materials for coating prostheses
- A61L27/34—Macromolecular materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- C—CHEMISTRY; METALLURGY
- C25—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES; APPARATUS THEREFOR
- C25D—PROCESSES FOR THE ELECTROLYTIC OR ELECTROPHORETIC PRODUCTION OF COATINGS; ELECTROFORMING; APPARATUS THEREFOR
- C25D11/00—Electrolytic coating by surface reaction, i.e. forming conversion layers
- C25D11/02—Anodisation
- C25D11/26—Anodisation of refractory metals or alloys based thereon
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/204—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials with nitrogen-containing functional groups, e.g. aminoxides, nitriles, guanidines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
- A61L2300/606—Coatings
Abstract
The invention relates to the technical field of dental implant coatings, in particular to a dental implant PDA-D-amino-TNT composite coating and a preparation method thereof. The method comprises the following steps: (1) preparing an anodic oxidation TNT array; (2) biological modification of anodized TNT arrays: preparing D-amino K122-4 polypeptide; soaking the test piece subjected to anodic oxidation reaction in a Tris solution of dopamine, and gently oscillating; taking out the test piece, washing with deionized water, and air-drying; immersing the dried test piece in a D-amino K122-4 polypeptide solution; taking out the test piece, washing with deionized water, and air-drying; d-amino K122-4 biological polypeptide is connected to an anodic oxidation TNT array through a polydopamine film interlayer; all test pieces were sterilized by ultraviolet irradiation.
Description
Technical Field
The invention relates to the technical field of dental implant coatings, in particular to a dental implant PDA-D-amino-TNT composite coating and a preparation method thereof.
Background
At present, dental titanium implants have been widely used in clinic, but implant failure caused by long implant-bone integration time and poor integration still occurs, and rapid and stable bone integration is one of important targets for implant material research.
In order to further increase the bioactivity of the implant, researchers have performed various modifications to the surface of titanium implants in order to obtain the desired osseointegration. Wherein, tiO 2 The Nano Tube (TNT) array structure is very similar to the cross section structure of the nano bone collagen, and from the bionics perspective, the ideal bone combination is more expected to be obtained through the regulation and control of the nano morphology structure of the implant surface on the bone tissue and the cell function. Therefore, it is one of the research hot spots for surface modification of titanium implants in recent years. Anodic oxidation is an economical and effective technique for preparing nano-structures on the surface of titanium, and a highly ordered TNT array with controllable morphology can be prepared on the surface of titanium by changing reaction conditions. The TNT array regulates the adhesion, proliferation and differentiation functions of various cells (mesenchymal stem cells, hematopoietic stem cells, endothelial cells, osteoblasts, osteoclasts and the like); at the same time, the size and surface characteristics (hydrophilicity, crystal structure and the like) of TNT have important influence on the cell function; in addition, due to the hollow tubular shape, TNT can also be used as a drug carrier to load and release substances such as growth factors, antibiotics and the like to the surrounding local bone tissue microenvironment, so that the bone-combining capacity of the traditional implant is further improved. Although TNT arrays have been widely studied to promote the binding of soft and hard tissues around implants, immune regulation and other biological activities, TNT arrays on the surfaces of dental implants have the defects of insufficient mechanical strength, weak binding force with titanium substrates, easy falling-off and the like, and the mechanical properties of TNT arrays still need to be further optimized.
Key factors affecting the morphology and mechanical properties of the TNT array include: (1) electrolyte properties: water content, fluoride ion concentration, degree of aging; (2) anodic oxidation reaction time; (3) anodic oxidation reaction voltage; (4) humidity and temperature controllability of an anodic oxidation reaction system; (5) a sterilization method, etc.
Heat treatment in an acetylene atmosphere, patrik et al found that TNT could be converted to semi-metallic oxycarbide (TiOxCy)/carbide (TiC). As the carbide content increases and the temperature increases, the hardness, tensile strength and friction properties of the TNT array are significantly enhanced. Although studies indicate that TiC and TiOxCy are well biocompatible, the effect of changes in chemical composition on TNT array bioactivity is not yet clear.
Alves et al have found that coating the TNT array surface with a hydroxyapatite coating enhances the binding strength of the TNT array, and if further annealing treatment is performed, the crystallization degree of the hydroxyapatite is increased, ca and Ti ions diffuse to the oxide/metal interface, and the binding strength of the TNT array and the titanium substrate is further improved. However, this method may cause a change in physicochemical properties of the implant surface while improving the mechanical strength of the TNT array, and the influence on the bioactivity of the implant surface has yet to be studied.
Disclosure of Invention
The invention aims to provide a dental implant PDA-D-amino-TNT composite coating and a preparation method thereof.
In order to solve the technical problems, the application provides the following technical scheme:
a preparation method of a dental implant PDA-D-amino-TNT composite coating comprises the following steps: (1) preparing an anodic oxidation TNT array; (2) biological modification of anodized TNT arrays.
Preparing an ethylene glycol organic solution, and aging for 10 hours through an anodic oxidation reaction, wherein the reaction voltage is 75V, and the ethylene glycol organic solution is used as an anodic oxidation electrolyte;
and (3) adopting an anodic oxidation method, taking a titanium sheet as an anode, taking platinum as a cathode, and carrying out a reaction for 10min at a voltage of 60V to prepare the anodic oxidation TNT array test piece.
Wherein the glycol organic solution contains 0.3% NH4F and 1% deionized water.
Wherein, the step (2) specifically comprises the following steps: preparing D-amino K122-4 polypeptide by solid-phase polypeptide synthesis method, and purifying by reversed-phase high performance liquid chromatography; the polypeptide sequence is as follows: ACTSNADNKYLPKTCQT, as shown in SEQ ID NO. 1;
d-amino K122-4 polypeptide synthesis sequence: synthesized sequentially from the C-terminal to the N-terminal of the peptide chain.
1) The synthesis steps are as follows:
a. swelling of the resin: the protected threonine (fmoc-Thr (tbu) -OH) resin was weighed and poured into a reaction column, and N, N-Dimethylformamide (DMF) was added for soaking for 30 minutes, and the mixture was drained.
b. Deprotection: an appropriate amount of deprotected solution (20% piperidine +80% dmf) was added to the reaction column, stirred with nitrogen for 30 minutes, drained and repeated 6 times.
c. Weighing: weighing 3 times of molar quantity of protected amino acid, and weighing 2.85 times of molar quantity of benzotriazol-N, N, N ', N' -tetramethylurea Hexafluorophosphate (HBTU) shrink agent for standby;
d. feeding: adding the prepared protected amino acid and HBTU into a reaction column, adding N-methyl morpholine (NMM) organic base with the amount which is six times that of the resin, stirring with nitrogen and stirring for 30 minutes;
e. washing after reaction: pumping the solution in the reaction column, adding a proper amount of DMF for washing, pumping nitrogen for 2 minutes, and repeating the operation for 3 times;
f. and (3) detection: taking a proper amount (10-20) of resin into a small test tube, adding two drops of solution A, solution B and solution C (solution A: 80% phenol +20% absolute ethyl alcohol; solution B: redistilled pyridine; solution C: 5 g ninhydrin +100ML absolute ethyl alcohol). Heating in a dry heater for 3 minutes (110 ℃ C.). If the solution is blue after being taken out and the resin has impurities which are not transparent, the reaction is not complete and the reaction needs to be repeated again; if the color of the solution is yellowish, the resin is colorless and transparent, the reaction is complete, the next amino acid can be connected, the steps b-f are repeated until the last amino acid is connected, the sequence can be obtained, and finally the protecting group Fmoc on Fmoc-Ala-OH is removed, so that the N end is exposed;
g. washing and drying after synthesis: after the last amino acid was taken over and the deprotected and washed, the reaction column was drained, an appropriate amount of methanol was added, nitrogen sparged for 2 minutes, drained, and an appropriate amount of dichloromethane solvent (DCM) was added, nitrogen sparged for 2 minutes, and the draining was repeated 3 times. Finally, adding a proper amount of methanol, stirring for 2 minutes by nitrogen, pumping and repeating for 2 times, and putting the resin into a proper vessel for vacuum drying for 12 hours for cutting.
(2) Cleavage of the polypeptide:
a. cutting: the dried resin was placed in a suitable round bottom flask and a suitable amount of the prepared cutting fluid (1 g/10ml,87.5% trifluoroacetic acid+5) was added% of phenylsulfide+2.5% of phenol+2.5% of 1, 2-ethanedithiol+2.5% of H 2 O), placing in a constant temperature shaking table, and oscillating for 2 hours at a constant temperature of 25 ℃;
b. filtering, namely filtering out resin particles by using a 50mL sand core funnel, pouring the filtrate into a glass container of a centrifuge tube, adding anhydrous diethyl ether with the volume of 6-8 times, and stirring while adding, wherein the precipitated white solid is the crude product of the required polypeptide;
c. washing: packaging the white solid into a centrifuge tube, sealing the centrifuge tube, putting the centrifuge tube into the centrifuge, centrifuging for 3 minutes at the rotating speed of 4000r/min, taking out, pouring out supernatant, adding diethyl ether, uniformly stirring by using a glass rod, centrifuging again, and repeating for 5 times;
d. and (3) drying: the polypeptide washed 5 times was placed in a vacuum dryer and vacuum dried for 24 hours. The obtained white powder is the crude product of the required polypeptide, and is weighed and purified.
(3) Purification of the polypeptide:
a. firstly, 10-100% analysis of the crude product is carried out, and after the retention time of a main peak is determined, linear gradient analysis is carried out;
b. solubility of test sample: water and acetonitrile (4:1) can fully dissolve the polypeptide;
c. dissolving 200mg polypeptide in 10ml dissolving bottle, ultrasonic dissolving, and filtering with 0.45um organic phase filter to obtain clear solution;
d. preparing a peak by linear gradient, and determining the alignment of the chromatograph;
e. tracking and feeding back the purity of the received liquid to obtain the liquid quantity meeting the purity requirement;
f. concentrating the collected qualified liquid at 38 ℃ to 50-100ml (direct freezing without concentration under special conditions);
g. the concentrated liquid was placed in a 100ml beaker, and frozen in a solid state with liquid nitrogen or dry ice, and dried.
Soaking the test piece prepared in the step (1) in 2mg/ml dopamine Tris solution, wherein the pH is 8.5, and gently oscillating for 12 hours; taking out the test piece, washing with deionized water for 3 times, and air-drying; the dried test piece is soaked in 1mg/ml D-amino K122-4 polypeptide solution, the pH=8.5, and the test piece is gently vibrated for 12 hours; taking out the test piece, washing with deionized water for 3 times, and air-drying; d-amino K122-4 biological polypeptide is connected to an anodic oxidation TNT array through a polydopamine film interlayer; all test pieces were sterilized by irradiation with ultraviolet rays for 2 hours.
Compared with the prior art, the PDA-D-amino-TNT composite coating of the dental implant and the preparation method thereof have the following beneficial effects:
the preparation method of the invention modifies the TNT array on the surface of the dental implant from the perspective of biomechanical property, thereby obtaining the implant surface coating structure with high mechanical strength and soft tissue compatibility around the implant.
According to the invention, a TNT array with stable mechanical property is prepared in an optimized anodic oxidation reaction system, and the D-amino K122-4 biological polypeptide is connected to the TNT array through the polydopamine film interlayer, so that the elastic modulus and hardness of the composite coating can be further improved.
The PDA-D-amino-TNT composite coating prepared by the invention has remarkable antibacterial property on dominant bacteria around the implant; has promoting effect on growth of main cells of implant passing through gingiva, human gingival fibroblast.
The PDA-D-amino-TNT composite coating for dental implant and the preparation method thereof are further described below with reference to the accompanying drawings.
Drawings
FIG. 1 is a schematic illustration of an anodic oxidation reaction.
FIG. 2 is a schematic diagram of the structure of an anodized TNT array; (a) the voltage is 60V, the TNT pipe diameter is about 50nm, and the structure is complete; (b) the voltage is 70V, the TNT pipe diameter is about 60nm, and cracks are seen under the mirror; (c) The voltage was 80v, the tnt portion peeled off, and cracks were visually generated.
FIG. 3 is an XRD pattern before and after annealing, with the arrow indicating the anatase diffraction peaks that occur after annealing.
FIG. 4 is a schematic diagram of the loading process of D-amino K122-4.
FIG. 5 shows XPS results; (A) TNT, TNT group surface O1S, ti2P, C S peak was most pronounced; (B) PDA-TNT, showing that N1S peak appears after PDA coating, and C1S peak rises; (C) The S2P peak appears after the grafting of the PDA-D-amino-TNT and the N1S peak rises.
FIG. 6 is a graph showing the proliferation of human gingival fibroblasts on the surfaces of four groups of test pieces.
Detailed Description
Abbreviations and key terms are defined as follows:
1. TiO2 nanotube (TNT)
2. Scanning Electron Microscope (SEM)
3. X-ray diffraction (XRD)
4. X-ray photoelectron spectroscopy (XPS)
5. Annealing TNT (Anatase TNT)
6. Dopamine loading TNT (PDA-TNT)
7. TNT loading of D-amino K122-4 (PDA-D-amino-TNT)
8. Streptococcus mutans (S.m)
9. Fusobacterium nucleatum (F.n)
10. Human Gingival Fibroblast (HGFs)
11. Titanium (Ti)
12. N, N-Dimethylformamide (DMF)
13. Phenyltrioxazole-N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HBTU)
14. N-methylmorpholine (NMM)
15. Dichloromethane (DCM)
(1) Optimizing an anodic oxidation reaction system and parameters to prepare the TNT array with stable structure.
Designing a bipolar electrochemical reaction system with a closed reaction environment, and preparing an ethylene glycol organic solution (containing 0.3% NH) 4 F+1% deionized water) and aged for 10 hours (reaction voltage 75V) by an anodic oxidation reaction as an anodic oxidation electrolyte.
The anodic oxidation method is adopted, titanium sheets are used as anodes, platinum is used as a cathode, the voltage range is 60-80V, and the reaction time is 10min (shown in figure 1). As the voltage increases, the pore size of the nanopore increases, and fig. 2 shows: when the voltage is 60V, the formed nano holes are orderly arranged along the micron-sized grooves on the titanium surface, the structure is complete, and no damage such as cracks, stripping and the like is seen; when the voltage is 70V, the formed nano holes are orderly arranged along the micron-sized grooves on the titanium surface, and when the amplification is 25K times, cracks are visible; at a voltage of 80V, the nanocoating was visible to the naked eye to crack or even spall. The higher the anodic oxidation voltage, the poorer the structural integrity and stability of the TNT array was demonstrated.
(2) Physical modification of anodized TNT arrays.
Heating the test piece prepared in the step (1) at 60V/10min to 450 ℃ in air at a heating rate of 4 ℃/min, and carrying out annealing treatment for 3 hours to change the crystal structure of the titanium dioxide: the XRD pattern in FIG. 3 shows strong diffraction peaks of anatase titania, indicating that TNT is converted from amorphous titania to anatase titania after heat treatment. SEM examination did not see TNT structural changes.
(3) Biological modification of anodic oxidation TNT array
The biological polypeptide D-amino K122-4 forms a self-assembled or folded form pilA Receptor Binding Domain (RBD) (disulfide ring formed by 128-144 amino acid residues coded by pseudomonas aeruginosa pilA gene), and the D-amino K122-4 has strong binding force with stainless steel metal, and chiral amino acid form can reduce sensitivity to protease, so that the biological polypeptide has good clinical application prospect.
The D-amino K122-4 polypeptide is prepared by adopting a solid-phase polypeptide synthesis method and is purified by reverse phase high performance liquid chromatography.
The polypeptide sequence is as follows: ACTSNADNKYLPKTCQT, as shown in SEQ ID NO. 1;
d-amino K122-4 polypeptide synthesis sequence: synthesized sequentially from the C-terminal to the N-terminal of the peptide chain.
1) The synthesis steps are as follows:
a. swelling of the resin: the protected threonine (fmoc-Thr (tbu) -OH) resin was weighed and poured into a reaction column, and N, N-Dimethylformamide (DMF) was added for soaking for 30 minutes, and the mixture was drained.
b. Deprotection: an appropriate amount of deprotected solution (20% piperidine +80% dmf) was added to the reaction column, stirred with nitrogen for 30 minutes, drained and repeated 6 times.
c. Weighing: weighing 3 times of molar quantity of protected amino acid, and weighing 2.85 times of molar quantity of benzotriazol-N, N, N ', N' -tetramethylurea Hexafluorophosphate (HBTU) shrink agent for standby;
d. feeding: adding the prepared protected amino acid and HBTU into a reaction column, adding N-methyl morpholine (NMM) organic base with the amount which is six times that of the resin, stirring with nitrogen and stirring for 30 minutes;
e. washing after reaction: pumping the solution in the reaction column, adding a proper amount of DMF for washing, pumping nitrogen for 2 minutes, and repeating the operation for 3 times;
f. and (3) detection: taking a proper amount (10-20) of resin into a small test tube, adding two drops of solution A, solution B and solution C (solution A: 80% phenol +20% absolute ethyl alcohol; solution B: redistilled pyridine; solution C: 5 g ninhydrin +100ML absolute ethyl alcohol). Heating in a dry heater for 3 minutes (110 ℃ C.). If the solution is blue after being taken out and the resin has impurities which are not transparent, the reaction is not complete and the reaction needs to be repeated again; if the color of the solution is yellowish, the resin is colorless and transparent, the reaction is complete, the next amino acid can be connected, the steps b-f are repeated until the last amino acid is connected, the sequence can be obtained, and finally the protecting group Fmoc on Fmoc-Ala-OH is removed, so that the N end is exposed;
g. washing and drying after synthesis: after the last amino acid was taken over and the deprotected and washed, the reaction column was drained, an appropriate amount of methanol was added, nitrogen sparged for 2 minutes, drained, and an appropriate amount of dichloromethane solvent (DCM) was added, nitrogen sparged for 2 minutes, and the draining was repeated 3 times. Finally, adding a proper amount of methanol, stirring for 2 minutes by nitrogen, pumping and repeating for 2 times, and putting the resin into a proper vessel for vacuum drying for 12 hours for cutting.
(2) Cleavage of the polypeptide:
a. cutting: the dried resin was placed in a suitable round bottom flask and a suitable amount of the prepared cutting fluid (1 g/10ml,87.5% trifluoroacetic acid +5% phenylsulfide +2.5% phenol +2.5% 1, 2-ethanedithiol +2.5% H) 2 O), placing in a constant temperature shaking table, and oscillating for 2 hours at a constant temperature of 25 ℃;
b. filtering, namely filtering out resin particles by using a 50mL sand core funnel, pouring the filtrate into a glass container of a centrifuge tube, adding anhydrous diethyl ether with the volume of 6-8 times, and stirring while adding, wherein the precipitated white solid is the crude product of the required polypeptide;
c. washing: packaging the white solid into a centrifuge tube, sealing the centrifuge tube, putting the centrifuge tube into the centrifuge, centrifuging for 3 minutes at the rotating speed of 4000r/min, taking out, pouring out supernatant, adding diethyl ether, uniformly stirring by using a glass rod, centrifuging again, and repeating for 5 times;
d. and (3) drying: the polypeptide washed 5 times was placed in a vacuum dryer and vacuum dried for 24 hours. The obtained white powder is the crude product of the required polypeptide, and is weighed and purified.
(3) Purification of the polypeptide:
a. firstly, 10-100% analysis of the crude product is carried out, and after the retention time of a main peak is determined, linear gradient analysis is carried out;
b. solubility of test sample: water and acetonitrile (4:1) can fully dissolve the polypeptide;
c. dissolving 200mg polypeptide in 10ml dissolving bottle, ultrasonic dissolving, and filtering with 0.45um organic phase filter to obtain clear solution;
d. preparing a peak by linear gradient, and determining the alignment of the chromatograph;
e. tracking and feeding back the purity of the received liquid to obtain the liquid quantity meeting the purity requirement;
f. concentrating the collected qualified liquid at 38 ℃ to 50-100ml (direct freezing without concentration under special conditions);
g. the concentrated liquid was placed in a 100ml beaker, and frozen in a solid state with liquid nitrogen or dry ice, and dried.
The specific loading process is as follows: immersing the test piece after the anodic oxidation reaction in 2mg/ml dopamine Tris (10 mM) solution, and gently oscillating for 12 hours at the pH of 8.5; taking out the test piece, washing with deionized water for 3 times, and air-drying; the dried test piece is soaked in 1mg/ml D-amino K122-4 polypeptide solution, the PH=8.5, and the test piece is gently vibrated for 12 hours; taking out the test piece, washing with deionized water for 3 times, and air-drying; d-amino K122-4 biological polypeptides were attached to TNT arrays via a polydopamine membrane (PDA) interlayer (FIG. 4). All test pieces were sterilized by irradiation with ultraviolet rays for 2 hours.
X-ray photoelectron spectroscopy (XPS) results (FIG. 5) showed that the N1S peak appeared after PDA coating and the C1S peak was elevated, the S2P peak appeared after grafting of the D-amino polypeptide and the N1S peak was elevated, demonstrating that the D-amino was stably bound to the TNT array. The content changes of Ti, O, C, N and S elements are shown in Table 1.
TABLE 1
(4) Influence of nano indentation method on TNT mechanical property compared with different modification methods
The nano indentation instrument G200 can realize the measurement of mechanical parameters such as material hardness, elastic modulus, contact stiffness and the like through indentation experiments. The contact stiffness can be obtained through the unloading curve, and the hardness and the elastic modulus of the maximum indentation can only be obtained in each compression cycle, so that the mechanical properties of the TNT group, the Anatase TNT group and the PDA-D-amino-TNT group are respectively analyzed and compared by taking the hardness and the elastic modulus of the coating as test indexes (see Table 2), the elastic modulus and the hardness of the Anatase TNT (annealed TNT) are not obviously enhanced after high-temperature annealing treatment, and the elastic modulus and the hardness of the composite coating are obviously improved after the TNT is modified by the PDA-D-amino.
TABLE 2
Because the annealing is processed by special large-scale equipment, the chemical composition of the surface of the annealed material can be changed, and the mechanical property of TNT is not obviously improved; the biological modification of grafting D-amino through the PDA interlayer is hopeful to become a new method for improving the biomechanical property of the TNT array.
(5) Colony counting method for evaluating antibacterial rate of implant surface structure against streptococcus mutans S.m and clostridium nucleatum F.n
Study the surface antibacterial property of the test piece is studied by selecting early-stage colonisation bacteria G+streptococcus mutans S.m UA159 on the surface of the implant and infection dominant bacteria G-Fusobacterium nucleatum F.n ATCC10953 around the implant.
The test pieces were divided into four groups, A being a polished Ti group, B being a TNT group, C being a PDA-TNT group, and D being a PDA-D-amino-TNT group. After two bacterial liquids are respectively mixed with four groups of test pieces for 24 hours, the test pieces are taken out, PBS is gently washed, the bacterial liquids are collected and inoculated in a solid culture medium, the culture is carried out until obvious bacterial colonies appear, the bacterial colonies are photographed, the bacterial colonies are counted, each group is repeated for 3 times, and the method is characterized in that: antibacterial ratio = control colony count-experimental colony count/control colony count, antibacterial ratio of experimental (B, C, D) was calculated. The antibacterial rates of the S.m strain on the surfaces of the four groups of test pieces are respectively as follows: 65.88%; c: -38.86%; d:81.52%. The antibacterial rates of the F.n strain on the surfaces of the four groups of test pieces are respectively as follows: 60 percent; c:44.2%; d:71.7%.
(6) Three parallel samples are set up on each of three test piece surfaces of TNT group, PDA-TNT group and PDA-D-amino-TNT group by CCK8 method, and absorbance values of cell suspension at 450nm and 630nm at 1 day, 2 day, 3 day and 4 day are detected respectively. Statistical results (fig. 6) show that PDA-D-amino-TNT group significantly promoted HGFs proliferation on days 3 and 4 of culture compared to TNT.
Therefore, the PDA-D-amino-TNT composite coating prepared by the invention has remarkable antibacterial property on dominant bacteria around the implant; and has promoting effect on growth of main cells of implant passing through gingiva, namely human gingival fibroblast.
The above examples are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (4)
1. A preparation method of a dental implant PDA-D-amino-TNT composite coating is characterized by comprising the following steps: the method comprises the following steps:
(1) Preparing an anodic oxidation TNT array:preparing an ethylene glycol organic solution, and aging for 10 hours through an anodic oxidation reaction, wherein the reaction voltage is 75V, and the ethylene glycol organic solution is used as an anodic oxidation electrolyte; the glycol organic solution contains 0.3% NH 4 F and 1% deionized water;
adopting an anodic oxidation method, taking a titanium sheet as an anode, taking platinum as a cathode, and carrying out a reaction for 10 minutes at a voltage of 60V to prepare an anodic oxidation TNT array test piece; heating the prepared test piece to 450 ℃ in air at a heating rate of 4 ℃/min, and carrying out annealing treatment for 3 hours;
(2) Biological modification of anodized TNT arrays: preparing a D-amino K122-4 polypeptide, wherein the polypeptide sequence is as follows: ACTSNADNKYLPKTCQT, as shown in SEQ ID NO. 1; soaking the test piece after anodic oxidation reaction in 2mg/ml dopamine Tris solution, wherein the pH value is 8.5, and gently oscillating for 12 hours; taking out the test piece, washing with deionized water for 3 times, and air-drying; the dried test piece is soaked in 1mg/ml D-amino K122-4 polypeptide solution, the pH=8.5, and the test piece is gently vibrated for 12 hours; taking out the test piece, washing with deionized water for 3 times, and air-drying; d-amino K122-4 biological polypeptide is connected to an anodic oxidation TNT array through a polydopamine film interlayer; all test pieces were sterilized by irradiation with ultraviolet rays for 2 hours.
2. The method for preparing the dental implant PDA-D-amino-TNT composite coating according to claim 1, characterized in that: in the step (2), the D-amino K122-4 polypeptide is prepared by adopting a solid-phase polypeptide synthesis method, and is purified by reverse-phase high performance liquid chromatography.
3. The method for preparing the dental implant PDA-D-amino-TNT composite coating according to claim 2, characterized in that: the synthesis sequence of the D-amino K122-4 polypeptide is as follows: sequentially synthesizing from the C end to the N end of the peptide chain; the specific method comprises the following steps:
(1) The synthesis steps are as follows:
a. swelling of the resin: weighing threonine resin with protection, pouring the threonine resin into a reaction column, adding N, N-dimethylformamide, soaking for 30 minutes, and pumping out;
b. deprotection: adding a proper amount of deprotection solution into a reaction column, introducing nitrogen, stirring and stirring for 30 minutes, pumping, and repeating for 6 times;
c. weighing: weighing 3 times of molar quantity of protected amino acid, and weighing 2.85 times of molar quantity of benzotriazol-N, N, N ', N' -tetramethyl urea hexafluorophosphate ester shrinking agent for standby;
d. feeding: adding the prepared protected amino acid and HBTU into a reaction column, adding N-methyl morpholine organic base with the amount which is six times that of the resin, stirring with nitrogen for 30 minutes;
e. washing after reaction: pumping the solution in the reaction column, adding a proper amount of DMF for washing, pumping nitrogen for 2 minutes, and repeating the operation for 3 times;
f. and (3) detection: taking a proper amount of resin in a small test tube, adding two drops of each of A, B and C, wherein the A is 80% phenol+20% absolute ethyl alcohol, the B is a heavy distilled pyridine, and the C is 5 g ninhydrin+100 ML absolute ethyl alcohol; heating in a dry heater for 3 minutes at 110 ℃; if the solution is blue after being taken out and the resin has impurities which are not transparent, the reaction is not complete and the reaction needs to be repeated again; if the color of the solution is yellowish, the resin is colorless and transparent, the reaction is complete, the next amino acid can be connected, the steps b-f are repeated until the last amino acid is connected, the polypeptide sequence can be obtained, and finally the protecting group Fmoc on Fmoc-Ala-OH is removed, so that the N end is exposed;
g. washing and drying after synthesis: after the last amino acid is connected and deprotected and washed, pumping, adding a proper amount of methanol into a reaction column, pumping for 2 minutes by nitrogen, adding a proper amount of dichloromethane solvent, pumping for 2 minutes by nitrogen, and repeating pumping for 3 times; finally adding a proper amount of methanol, stirring for 2 minutes by nitrogen, pumping and repeating for 2 times, and vacuum drying the resin in a proper vessel for 12 hours and cutting;
(2) Cleavage of the polypeptide:
a. cutting: putting the dried resin into a proper round bottom flask, adding a proper amount of prepared cutting liquid, and placing the mixture into a constant temperature shaking table to oscillate for 2 hours at a constant temperature of 25 ℃;
b. filtering, namely filtering out resin particles by using a 50mL sand core funnel, pouring the filtrate into a glass container of a centrifuge tube, adding anhydrous diethyl ether with the volume of 6-8 times, and stirring while adding, wherein the precipitated white solid is the crude product of the required polypeptide;
c. washing: packaging the white solid into a centrifuge tube, sealing the centrifuge tube, putting the centrifuge tube into the centrifuge, centrifuging for 3 minutes at the rotating speed of 4000r/min, taking out, pouring out supernatant, adding diethyl ether, uniformly stirring by using a glass rod, centrifuging again, and repeating for 5 times;
d. and (3) drying: placing the polypeptide washed 5 times into a vacuum dryer for vacuum drying for 24 hours; the obtained white powder is the crude product of the required polypeptide, and is weighed and purified;
(3) Purification of the polypeptide:
a. firstly, 10-100% analysis of the crude product is carried out, and after the retention time of a main peak is determined, linear gradient analysis is carried out;
b. solubility of the test sample; water and acetonitrile fully dissolve the polypeptide;
c. dissolving 200mg polypeptide in 10ml dissolving bottle, ultrasonic dissolving, and filtering with 0.45um organic phase filter to obtain clear solution;
d. preparing a peak by linear gradient, and determining the alignment of the chromatograph;
e. tracking and feeding back the purity of the received liquid to obtain the liquid quantity meeting the purity requirement;
f. concentrating the collected qualified liquid at low temperature at 38deg.C to 50-100 ml;
g. the concentrated liquid was placed in a 100ml beaker, and frozen in a solid state with liquid nitrogen or dry ice, and dried.
4. A dental implant PDA-D-amino-TNT composite coating prepared by the preparation method of any one of claims 1 to 3.
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| "Incorporating TiO2 nanotubes with a peptide of D-amino K122-4 (D) for enhanced mechanical and photocatalytic properties";L. Q. Guo等;《Sci. Rep.》;22247:1-9 * |
| "基于生物修饰的304不锈钢表面抗菌性能";曹攀 等;《中国表面工程》;第30卷(第3期);1-7 * |
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| CN116920173A (en) | 2023-10-24 |
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